RETRACTED ARTICLE: Biosynthesis of Zn-doped CuFe2O4 nanoparticles and their cytotoxic activity

Zn-doped CuFe2O4 nanoparticles (NPs) were eco-friendly synthesized using plant extract. These nanoparticles were characterized by X-ray diffraction, Fourier-transform infrared spectroscopy, scanning electron microscope (SEM), energy-dispersive X-ray spectroscopy and thermal gravimetric analysis (TGA). SEM image showed spherical NPs with size range less than 30 nm. In the EDS diagram, the elements of zinc, copper, iron, and oxygen are shown. The cytotoxicity and anticancer properties of Zn-doped CuFe2O4 NPs were evaluated on macrophage normal cells and A549 lung cancer cells. The cytotoxic effects of Zn-doped CuFe2O4 and CuFe2O4 NPs on A549 cancer cell lines were analyzed. The Zn-doped CuFe2O4 and CuFe2O4 NPs demonstrated IC50 values 95.8 and 278.4 µg/mL on A549 cancer cell, respectively. Additionally, Zn-doped CuFe2O4 and CuFe2O4 NPs had IC80 values of 8.31 and 16.1 µg/mL on A549 cancer cell, respectively. Notably, doping Zn on CuFe2O4 NPs displayed better cytotoxic effects on A549 cancer cells compared with the CuFe2O4 NPs alone. Also spinel nanocrystals of Zn-doped CuFe2O4 (~ 13 nm) had a minimum toxicity (CC50 = 136.6 µg/mL) on macrophages J774 Cell Line.

www.nature.com/scientificreports/ cancer cells necrosis (in vitro) by increasing the oxidative stress and caspase-3 activity 1 . Also, these multimetallic magnetic particles have low production costs, and can be recycled in water treatment 90,93 . Magnetic zinc ferrites (ZnFe 2 O 4 ) are recyclable and biocompatible catalysts with high anti-inflammatory activity 94 . Zinc ferrite NPs demonstrated good biocompatibility and hemocompatibility with human dermal fibroblast cells (HDF) and red blood cells (RBC), respectively. On the other hand, they have high toxicity against Gram-positive and Gram-negative bacteria by increasing reactive oxygene stress (ROS) 95 . Ferrite multi-metals such as nickel zinc ferrite and chromium copper ferrite have shown promising clinical and biomedical applicability due to their unique physicochemical features. The antibacterial properties of chromium copper ferrite NPs are greater than those of copper ferrite NPs. With the addition of chromium metal, the surface-to-volume ratio in chromium copper ferrite NPs was increased, and these NPs had more damaging activity against bacterial membranes 96 . In vitro studies demonstrated that nickel zinc ferrite NPs had time-dependent and concentration cytotoxicity against colon HT29, breast MCF7, and liver HepG2 cancer cells. They could increase the apoptosis of cancer cells by mitochondrial and chromosomal damages. Maximum cell death in liver cancer cells was at a concentration of 100 µg/mL, and also it was observed in colon and breast cancer cells at a concentration of 1000 µg/mL 97 .
Plant-mediated synthesis of Zn-doped CuFe 2 O 4 NPs. The young leaves of the Nasturtium plant were washed with deionized water. The surface moisture of the leaves was removed at 27 °C and turned into a soft powder. 1 g of plant powder was mixed by 10 mL of deionized water and stirred at room temperature for 24 h. The plant extract was filtered by Whatman filter paper (the size No. 40) and centrifuged. Fe(NO 3 ) 3 ·9H 2 O (1.7 g), Zn(NO 3 ) 2 ·6H 2 O (0.8 g), and CuCl 2 ·2H 2 O (0.8 g) salts were added to 21 mL of plant extract and dissolved at room temperature under vigorous stirring, respectively. After complete dissolution of salts, the pH of the mixture was increased from 4 to 7 by adding NaOH 1 M under the same conditions. After that, 15 mL of deionized water was added dropwise to the mixture and sterilized continuously for 2 h at room temperature. The resulting mixture was transferred to an autoclave and placed in an oven at 170 °C for 13 h. The synthesized NPs were washed several times with deionized water. Finally, the obtained powder was dried at 80 °C for 10 h and calcined at 400 °C for 10 h.

Cytotoxic effects of Zn-doped CuFe 2 O 4 NPs on macrophages J774 cell line.
For the cytotoxicity analysis of NPs on macrophages J774 cell line, we determined the CC 50 (cytotoxicity concentration for 50% of cells) for various concentrations (1, 5, 10, 50, 100, 500, and 1000 µg/mL) of Zn-doped CuFe 2 O 4 , ZnO 98 , CuO 99 , and CuFe 2 O 4 NPs on macrophages. Macrophage cells were plated at 10 6 cells/mL in 96-well Lab-Tek (Nunc, USA) and left to adhere for 24 h at 37 °C and 5% CO 2 . After removing the non-adherent cells by washing with DMEM medium, the cells were incubated at similar conditions as mentioned before. Thereafter, 190 µL of complete DMEM medium was added in each well, and after that 10 µL of NPs dilution was added (as previously prepared in medium). Macrophages were preserved with the NPs from 1 to 1000 µg/mL for 72 h. The cytotoxicity rate was evaluated using the WST1 colorimetric cell viability assay as previously defined in the promastigote sensitivity assay. All experiments were performed in triplicate similar to the previous stages 100 .

Cytotoxicity analysis of Zn-doped CuFe 2 O 4 NPs against cancer cells. The cytotoxicity of Zn-
doped CuFe 2 O 4 , ZnO, CuO, and CuFe 2 O 4 NPs (various concentrations: 1, 5, 10, 50, 100, 500, and 1000 µg/mL) against A549 lung cancer cells was measured based on MTT assay for 72 h. 10 4 cells/cm 2 were seeded in 96-well plates. After attaching the cells to the plate wall, different concentrations of NPs were added and incubated at 37 °C with 5% CO 2 for 72 h. After this procedure, the cells were washed with phosphate buffer saline (PBS), and the medium was discarded. In the following, 5 mg/mL of MTT dye in PBS was applied to each well, and the plate was incubated for 4 h. 100 µL of DMSO solution was added to each well, and then stored in the dark place at 25 °C for 15 min. Finally, using a microplate reader, the absorbance of dissolved formazan was measured at 570 nm (DYNEX MRX, USA). The proportion of viable cells to untreated cells was deployed to characterize the

Results
The XRD analysis was performed using an X'PertPro (Panalytical Company, Holland) diffractometer with wavelength of X-ray beam 1.5 Å and Cu anode material. XRD measurements were performed to determine the crystalline phase and nature of biogenic nanostructures (2θ range from 10° to 80°). XRD data of plant extract and nanostructures are depicted in Fig. 1a In the XRD pattern, the reflection (311) is the most intense peak. The lattice constant was calculated using the interplanar spacing distance and the respective (hkl) parameters using the following relation 105 : The crystallite size was estimated from the most intense peak of XRD data (311). The crystallite size was calculated as a function of Zn content x using Debye-Scherrer's formula (D = 0.9λ/β cos θ). In this formula "λ" is the wavelength of the X-ray radiation, "β" is the full-width half maximum and "2θ" is the diffraction angle. As a result, the crystallite size of NPs was found to be ~ 20 nm.
FTIR analysis of Zn-doped CuFe 2 O 4 NPs in the range of 300 to 4000 cm −1 with KBr pellet was performed by tensor II (Bruker Company, Germany) device. FTIR analysis identified the functional groups and chemical bonds present in the synthesized NPs (Fig. 2 Fig. 5a, respectively. According to CC 50 values, Zn-doped CuFe 2 O 4 , ZnO, and CuFe 2 O 4 NPs displayed no significant cytotoxic effects against macrophage cells, but CuO NPs illustrated significant cytotoxic effects against normal macrophage cells. Based on our results, Zn-doped CuFe 2 O 4 , ZnO, and CuFe 2 O 4 NPs were safer for mammalian cells. According to the results, CuO NPs caused oxidative stress and genetic toxicity in mammalian normal cells 113,114 . The cytotoxic effects of Zn-doped CuFe 2 O 4 , ZnO, CuO, and CuFe 2 O 4 NPs exposed to 1-1000 µg/mL on A549 cancer cell lines are shown in Fig. 5b ) is an aquatic plant that has significant amounts of iron, calcium, folic acid, glucosinolates, and vitamins C and A. This medicinal plant has significant anticancer and antioxidant properties due to its phenolic compounds 118 . Methanolic extract of this plant has been shown to increase A549 cancer cell mortality by activating apoptotic agents 118 . On the other hand, multimetallic NPs have been focused by researchers due to the synergy of metal elements and multifunctionality 119,120 . Additionally, by increasing the phenolic compounds of Nasturtium extract, the antioxidant activity was enhanced with the lowest IC 50 121 .

Conclusion
Zn-doped CuFe 2 O 4 nanopowders were successfully synthesized in one step using Nasturtium plant extract. The NPs were characterized by XRD, FTIR, EDS, TGA, and SEM. The biocompatibility and cytotoxicity of Zn-doped CuFe 2 O 4 NPs were evaluated on macrophages cell Line. Additionally, the anticancer properties of Zn-doped CuFe 2 O 4 NPs against A549 lung cancer cells were evaluated. As a result, doping Zn on CuFe 2 O 4 NPs displayed better cytotoxic effects on A549 cancer cells compared with the CuFe 2 O 4 NPs alone. Also spinel crystallites of Zn-doped CuFe 2 O 4 (~ 13 nm) had a minimum toxicity (CC 50 = 136.6 µg/mL) on macrophages J774 Cell Line. The Zn-doped CuFe 2 O 4 are multi-metallic with suitable applicability and biocompatibility, which should be further studied particularly for the treatment and diagnosis of cancers and infectious diseases. Additionally, these nanomaterials with unique optical and magnetic properties can be considered as attractive candidates for catalytic applications.

Data availability
The datasets used and analysed during the current study available from the corresponding author on reasonable request.